The ephrins are plasma membrane associated proteins that send signals to their target cells via a family of transmembrane receptor tyrosine kinases called the Ephs. Mammalian ephrins regulate many processes during animal development that involve cell-cell interactions including boundary formation during organogenesis, blood vessel formation, neural crest cell migration, and the topographic guidance of axons. Our studies suggest a new role for ephrinBs in the development of central nervous system synapses. We found that binding of ephrinB to its cognate receptor EphB on the surface of neurons promotes the association of the ephrinB/EphB complex with the N-methyl D-aspartate sub-type of glutamate receptor, a receptor that has been implicated in synapse development and plasticity. The interaction of EphB with NMDA receptors promotes calcium influx into neurons leading to the activation of a gene transcription program that may promote synapse development. Our characterization of the mechanism of ephrinB/EphB potentiation of NMDA receptor function revealed that ephrinB treatment leads to tyrosine phosphorylation of the NMDA receptor NR2B subunit by a Src family tyrosine kinase. These findings raise several questions that form the basis of this proposal. One question is what is the importance of the EphB/NMDA receptor interaction for nervous system development. Additional questions concern the specific mechanisms that mediate ephrinB modulation of NMDA receptors. We propose the following specific aims to address these issues: 1) to potentiate or perturb the formation of the EphB/NMDA receptor complex and assess the effect on NMDA receptor signaling. 2) to identify the sequences within the NMDA receptor that mediate the interaction with EphB and to use this information to develop knock-in mice that express NMDA receptors that cannot interact with EphBs. 3) to determine the mechanisms by which ephrinB/EphB potentiated NMDA receptors activate gene transcription. We will reconstitute NMDA receptor signaling in neurons from mice that lack NMDA receptors and by mutagenesis characterize the pathways that convey the EphB/NMDA receptor signal with cells. These experiments will provide insight into the importance of ephrins and Ephs for nervous system development and may reveal how defects in this Process lead to neural degeneration or oncogenesis